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6 years ago in Molecular Biology , Synthetic Biology By Payal Homraj Bhagat

How is molecular cloning performed in modern molecular biology?

The fundamentals of cutting, pasting, and amplifying DNA remain, but the tools have evolved dramatically from older manuals. As a researcher returning to the bench, I want to understand the modern, efficient pipeline—is it all Gibson Assembly and Golden Gate now, or do restriction enzymes still have a place?

Modernism Cloning

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By Govind Answered 5 years ago

In modern labs, the workflow is dominated by sequence-independent, in vitro assembly methods like Gibson Assembly or Golden Gate. I’ve largely moved away from traditional restriction/ligation for standard constructs. The process now is: design fragments with 20-40bp overlaps in silico, order them as gBlocks or amplify via PCR, and perform a one-pot, isothermal assembly reaction. Transformation and colony PCR verification remain constants. The revolution is in the design freedom and speed; what took weeks now often takes days. Restriction enzymes are now mostly for diagnostics, not construction.

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